Mechanical forces influence a multitude of fundamental cellular processes, yet little is known about the mechanism. It is known that integrin receptors and focal adhesions are the nexus for mechano-communication and that input from the acto-myosin contractility mechanism is needed, although the specifics are still lacking. Calpains are an ideal regulator of this communication system because they localize to and proteolyze focal adhesion proteins and interfere with the production of traction forces (inside out forces) and the sensing of external mechanical forces (outside-in). The objective of this application is to determine the mechanism by which calpains regulate traction force and mechanosensing. The central hypothesis is that the small subunit calpain 4 is a key regulator of mechanosensing and traction force production, functioning independent of the catalytic activity of the large subunits, calpain 1 and 2, in traction force production and in conjunction with them for mechanosensing. Guided by strong preliminary data, this hypothesis will be tested by pursuing the following specific aims: 1) Determine the structural domains of the Calpain molecules responsible for the production of traction forces and mechanosensing;2) Identify the role of the small GTPases and tyrosine phosphorylation in this regulatory scheme;and 3) Determine how focal adhesion/stress fiber interactions are regulated by calpains for force production and mechanosensing. These aims will be addressed by using a combination of molecular, biochemical, imaging and biophysical assays. These assays include site-directed mutagenesis, 2-D gel electrophoresis, and live cell imaging using TIRF and traction force microscopy (TFM) along with physical micromanipulations. Results from this study are expected to have a positive impact on the fields of calpain proteases and mechanobiology. RELEVANCE TO PUBLIC HEALTH: Understanding the role of calpains in mechanical forces could ultimately lead to therapeutic approaches to target a number of diseases as well as impact the field of tissue bioengineering.Narrative Migration and mechanical forces influence wound healing, development and cancer metastasis. The Calpain proteases have been implicated in these processes and numerous diseases ranging from cancer to arthritis. Given the number of diseases affected by calpains and the broad implications of traction forces and mechanosensing, it is imperative that we understand the roles of calpains in the production of traction forces and mechanosensing potentially resulting in novel therapeutic targets for these diseases and for use as tissue engineering or biological tools.